Method of evaluating inflammatory skin disorders using IL-17 and/or IL-19

ABSTRACT

Provided are methods for diagnosing the propensity of a subject to develop skin inflammation, in particular, psoriasis. Also provided are methods of treatment with antagonists of IL-17, IL-19, and/or IL-23.

This application is a Continuation application of U.S. patentapplication Ser. No. 11/218,446, filed Sep. 2, 2005 now abandoned, whichis a Continuation-In-Part application of U.S. patent application Ser.No. 11/120,518, filed May 2, 2005, which claims benefit from U.S.Provisional Patent Application No. 60/567,747, filed May 3, 2004.

FIELD OF THE INVENTION

The present invention relates to methods of analysis the propensity todevelop skin inflammatory disorders, in particular, psoriasis. Alsoprovided is a method of treatment to prevent such skin inflammatorydisorders using antagonists of IL- 17A, IL-17F, IL-19, and/or IL-23.

BACKGROUND OF THE INVENTION

The skin serves as an important boundary between the internal milieu andthe environment, preventing contact with potentially harmful antigens.In the case of antigen/pathogen penetration, an inflammatory response isinduced to eliminate the antigen. This response leads to a dermalinfiltrate that consists predominantly of T cells, polymophonuclearcells, and macrophages (see, e.g., Williams and Kupper (1996) Life Sci.,58:1485-1507.) Normally, this inflammatory response, triggered by thepathogen, is under tight control and will be halted upon elimination ofthe pathogen.

In certain cases, this inflammatory response occurs without externalstimuli and without proper controls, leading to cutaneous inflammation.Cutaneous inflammation, the result of the cellular infiltrate notedabove as well as the secreted cytokines from these cells, encompassesseveral inflammatory disorders such as cicatricial pemphigoid,scleroderma, hidradenitis suppurativa, toxic epidermal necrolysis, acne,osteitis, graft vs. host disease (GvHD), pyroderma gangrenosum, andBehcet's Syndrome (see, e.g., Willams and Griffiths (2002) Clin. Exp.Dermatol., 27:585-590). The most common form of cutaneous inflammationis psoriasis.

Psoriasis is characterized by T cell mediated hyperproliferation ofkeratinocytes coupled with an inflammatory infiltrate. The disease hascertain distinct overlapping clinical phenotypes including chronicplaque lesions, skin eruptions, and pustular lesions (see, e.g.,Gudjonsson, et al. (2004) Clin Exp. Immunol. 135:1-8). Approximately 10%of psoriasis patients develop arthritis. The disease has a strong butcomplex genetic predisposition, with 60% concordance in monozygotictwins.

The typical psoriatic lesion is a well defined erythematous plaquecovered by thick, silvery scales. The inflammation andhyperproliferation of psoriatic tissue is associated with a differenthistological, antigenic, and cytokine profile than normal skin. Amongthe cytokines associated with psoriasis are: TNFα, IL-19, IL-18, IL-15,IL-12, IL-7, IFNγ, IL-17A and IL-23 (see, Gudjonsson, et al., supra).IL-17A has been detected in psoriatic skin.

To date, prediction of psoriasis flare-ups has been hampered by lack ofknowledge of the cytokine changes between nonlesional and lesionalpsoriatic tissue. The present invention fills this unmet need byproviding a method of comparing IL-17A, IL-17F, and IL-19 expression innonlesional psoriatic skin relative to normal skin, thus affording theability to assess probability of the formation of lesional psoriasis.

SUMMARY OF THE INVENTION

The invention is based, in part, upon the discovery that IL-17A, IL-17F(collectively, “IL-17”), and IL-19 mRNA levels are comparably elevatedin non-lesional psoriatic tissue as compared to normal or non-psoriaticskin tissue.

The present invention provides a method of evaluating the propensity ofa subject to develop an inflammatory skin disorder comprising: a)obtaining a sample of skin from the subject; and b) quantifying thelevel of IL-17 or IL-19 expression in the sample. In one embodiment, theIL-17 or IL-19 expression is mRNA expression, and level of IL-17 orIL-19 expression is quantified by real-time PCR.

The invention further provides that the inflammatory skin disorder iscutaneous inflammation, in particular, psoriasis. The skin sample can befrom lesional or non-lesional psoriatic skin. In certain embodiments,the level of IL-17 expression in the skin sample is an average valuebetween 5 and 20 fold higher in than normal skin. The average value forIL-19 expression can be between 5 and 130 fold higher than normal skin.

In a further embodiment, the skin sample is from non-lesional psoriaticskin. Also encompassed is that the non-lesional psoriatic skin sample isfrom a subject having: a) a family history of psoriasis; or b)previously presented psoriatic symptoms. In a further embodiment,subject is a human.

The present invention also provides a method of preventing skininflammation comprising administering to a subject exhibiting apropensity to develop skin inflammation: a) an antagonist of IL-17; b)an antagonist of IL-23; c) an antagonist of IL-19; or d) an antagonistof at least two cytokines selected from the group consisting of IL-17,IL-19, or IL-23. In one embodiment the skin inflammation is cutaneousinflammation, in particular, psoriasis. In this method of preventingskin inflammation, subject expresses an average value of at least 5 foldhigher IL-17 expression in a non-lesional psoriatic skin sample comparedto a normal skin sample, as quantified by real-time PCR. The averagevalue of IL-19 expression is at least 20 fold higher.

The invention further provides that the antagonist of IL-17, IL-19,and/or IL-23 is an: a) antibody or binding fragment thereof; b) siRNA;or c) a small molecule inhibitor. In a further embodiment, the antibodyis: a) a polyclonal antibody; b) a monoclonal antibody; c) a humanizedantibody; d) a bi-specific antibody.

DETAILED DESCRIPTION

As used herein, including the appended claims, the singular forms ofwords such as “a,” “an,” and “the” include their corresponding pluralreferences unless the context clearly dictates otherwise. All referencescited herein are incorporated by reference to the same extent as if eachindividual publication, patent application, or patent, was specificallyand individually indicated to be incorporated by reference.

I. Definitions.

“Activity” of a molecule may describe or refer to binding of themolecule to a ligand or to a receptor, to catalytic activity, to theability to stimulate gene expression, to antigenic activity, to themodulation of activities of other molecules, and the like. “Activity” ofa molecule may also refer to activity in modulating or maintainingcell-to-cell interactions, e.g., adhesion, or activity in maintaining astructure of a cell, e.g., cell membranes or cytoskeleton. “Activity”may also mean specific activity, e.g., [catalytic activity]/[mgprotein], or [immunological activity]/[mg protein], or the like.

“Administration” and “treatment,” as it applies to an animal, human,experimental subject, cell, tissue, organ, or biological fluid, refersto contact of an exogenous pharmaceutical, therapeutic, diagnosticagent, or composition to the animal, human, subject, cell, tissue,organ, or biological fluid. “Administration” and “treatment” can refer,e.g., to therapeutic, pharmacokinetic, diagnostic, research, andexperimental methods. Treatment of a cell encompasses contact of areagent to the cell, as well as contact of a reagent to a fluid, wherethe fluid is in contact with the cell. “Administration” and “treatment”also means in vitro and ex vivo treatments, e.g., of a cell, by areagent, diagnostic, binding composition, or by another cell. Treatmentencompasses methods using a purified immune cell, e.g., in a mixed cellreactions or for administration to a research, animal, or human subject.The invention contemplates treatment with a cell, a purified cell, astimulated cell, a cell population enriched in a particular cell, and apurified cell. Treatment further encompasses situations where anadministered reagent or administered cell is modified by metabolism,degradation, or by conditions of storage.

“Amino acid” refers to naturally occurring and synthetic amino acids, aswell as amino acid analogs and amino acid mimetics that function in amanner similar to the naturally occurring amino acids. Naturallyoccurring amino acids are those encoded by the genetic code, includingselenomethionine, as well as those amino acids that are modified afterincorporation into a polypeptide, e.g., hydroxyproline, O-phosphoserine,O-phosphotyrosine, gamma-carboxyglutamate, and cystine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an α-carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) ormodified peptide backbones, but retain the same basic chemical structureas a naturally occurring amino acid. Amino acid mimetic refers to achemical compound that has a structure that is different from thegeneral chemical structure of an amino acid, but that functions in amanner similar to a naturally occurring amino acid. Amino acids may bereferred to herein by either their commonly known three letter symbolsor by their one-letter symbols.

“Binding composition” refers to a molecule, small molecule,macromolecule, antibody, a fragment or analogue thereof, or solublereceptor, capable of binding to a target. “Binding composition” also mayrefer to a complex of molecules, e.g., a non-covalent complex, to anionized molecule, and to a covalently or non-covalently modifiedmolecule, e.g., modified by phosphorylation, acylation, cross-linking,cyclization, or limited cleavage, which is capable of binding to atarget. “Binding composition” may also refer to a molecule incombination with a stabilizer, excipient, salt, buffer, solvent, oradditive, capable of binding to a target. “Binding” may be defined as anassociation of the binding composition with a target where theassociation results in reduction in the normal Brownian motion of thebinding composition, in cases where the binding composition can bedissolved or suspended in solution.

“Bispecific antibody” generally refers to a covalent complex, but mayrefer to a stable non-covalent complex of binding fragments from twodifferent antibodies, humanized binding fragments from two differentantibodies, or peptide mimetics derived from binding fragments from twodifferent antibodies. Each binding fragment recognizes a differenttarget or epitope, e.g., a different receptor, e.g., an inhibitingreceptor and an activating receptor. Bispecific antibodies normallyexhibit specific binding to two different antigens.

“Cutaneous Inflammation” refers to improper regulation of the immuneresponse in the skin or dermis, leading to an infiltrate of inflammatorycells and release of various inflammatory factors, including cytokines.Cutaneous inflammation includes psoriasis, atopic dermatitis,scleroderma, and the like.

Endpoints in activation or inhibition can be monitored as follows.Activation, inhibition, and response to treatment, e.g., of a cell, skintissue, keratinocyte, physiological fluid, tissue, organ, and animal orhuman subject, can be monitored by an endpoint. The endpoint maycomprise a predetermined quantity or percentage of, e.g., an indicia ofinflammation, oncogenicity, or cell degranulation or secretion, such asthe release of a cytokine, toxic oxygen, or a protease. The endpoint maycomprise, e.g., a predetermined quantity of ion flux or transport; cellmigration; cell adhesion; cell proliferation; potential for metastasis;cell differentiation; and change in phenotype, e.g., change inexpression of gene relating to inflammation, apoptosis, transformation,cell cycle, or metastasis (see, e.g., Knight (2000) Ann. Clin. Lab. Sci.30:145-158; Hood and Cheresh (2002) Nature Rev. Cancer 2:91-100; Timme,et al. (2003) Curr. Drug Targets 4:251-261; Robbins and Itzkowitz (2002)Med. Clin. North Am. 86:1467-1495; Grady and Markowitz (2002) Annu. Rev.Genomics Hum. Genet. 3:101-128; Bauer, et al. (2001) Glia 36:235-243;Stanimirovic and Satoh (2000) Brain Pathol. 10:113-126).

To examine the extent of inhibition, for example, samples or assayscomprising a given, e.g., protein, gene, cell, or organism, are treatedwith a potential activator or inhibitor and are compared to controlsamples without the inhibitor. Control samples, i.e., not treated withantagonist, are assigned a relative activity value of 100%. Inhibitionis achieved when the activity value relative to the control is about 90%or less, typically 85% or less, more typically 80% or less, mosttypically 75% or less, generally 70% or less, more generally 65% orless, most generally 60% or less, typically 55% or less, usually 50% orless, more usually 45% or less, most usually 40% or less, preferably 35%or less, more preferably 30% or less, still more preferably 25% or less,and most preferably less than 25%. Activation is achieved when theactivity value relative to the control is about 110%, generally at least120%, more generally at least 140%, more generally at least 160%, oftenat least 180%, more often at least 2-fold, most often at least 2.5-fold,usually at least 5-fold, more usually at least 10-fold, preferably atleast 20-fold, more preferably at least 40-fold, and most preferabl

“Exogenous” refers to substances that are produced outside an organism,cell, or human body, depending on the context. “Endogenous” refers tosubstances that are produced within a cell, organism, or human body,depending on the context.

“IL-17” as used herein, unless specifically stated otherwise, can meanIL-17A and/or IL-17F.

The “level of IL-17 expression” or “level of IL-19 expression” refers toreal-time PCR values that are normalized to ubiquitin. Kruskal-Wallisstatistical analysis is performed on log transformed data (medianmethod). Typically the calculated average level of IL-17 mRNA expressionin non-lesional psoriatic tissues is between 2 and 50 fold higher thannormal tissues. Preferably the level is between 5 and 20 fold higher.

A “marker” relates to the phenotype of a cell, tissue, organ, animal, orhuman subject. Markers are used to detect cells, e.g., during cellpurification, quantitation, migration, activation, maturation, ordevelopment, and may be used for both in vitro and in vivo studies. Anactivation marker is a marker that is associated with cell activation.

“Monofunctional reagent” refers, e.g., to an antibody, bindingcomposition derived from the binding site of an antibody, an antibodymimetic, a soluble receptor, engineered, recombinant, or chemicallymodified derivatives thereof, that specifically bind to a single type oftarget. For example, a monofunctional reagent may contain one or morefunctioning binding sites for an IL-17, IL-19, or IL-23 ligand orreceptor. “Monofunctional reagent” also refers to a polypeptide,antibody, or other reagent that contains one or more functioning bindingsites for, e.g., IL-17, IL-19, or IL-23 ligand or receptor and one ormore non-functioning binding sites for another type of receptor. Forexample, a monofunctional reagent may comprise an antibody binding sitefor IL-17, IL-19, or IL-23 ligand or receptor plus an Fc fragment thathas been engineered so that the Fc fragment does not specifically bindto Fc receptor.

“Nucleic acid” refers to deoxyribonucleotides or ribonucleotides andpolymers thereof in either single stranded or double-stranded form. Theterm nucleic acid may be used interchangeably with gene, cDNA, mRNA,oligonucleotide, and polynucleotide. A particular nucleic acid sequencealso implicitly encompasses “allelic variants” and “splice variants.”

“Condition” of skin encompasses disorders but also states that are notnecessarily classified as disorders, e.g., cosmetic conditions or statesof normal physiology. Disorders of a the skin encompass disorders of acell, where the cell is in the same genetic lineage of the skin, e.g., aprecursor cell of dermal keratinocytes where the precursor is committedto becoming a keratinocyte.

“Sample” refers to a sample from a human, animal, or to a researchsample, e.g., a cell, tissue, organ, fluid, gas, aerosol, slurry,colloid, or coagulated material. The “sample” may be tested in vivo,e.g., without removal from the human or animal, or it may be tested invitro. The sample may be tested after processing, e.g., by histologicalmethods. “Sample” also refers, e.g., to a cell comprising a fluid ortissue sample or a cell separated from a fluid or tissue sample.“Sample” may also refer to a cell, tissue, organ, or fluid that isfreshly taken from a human or animal, or to a cell, tissue, organ, orfluid that is processed or stored.

Small molecules are provided for the treatment of physiology anddisorders of the skin, e.g., cutaneous inflammation. “Small molecule” isdefined as a molecule with a molecular weight that is less than 10 kD,typically less than 2 kD, and preferably less than 1 kD. Small moleculesinclude, but are not limited to, inorganic molecules, organic molecules,organic molecules containing an inorganic component, moleculescomprising a radioactive atom, synthetic molecules, peptide mimetics,and antibody mimetics. As a therapeutic, a small molecule may be morepermeable to cells, less susceptible to degradation, and less apt toelicit an immune response than large molecules. Small molecule toxinsare described (see, e.g., U.S. Pat. No. 6,326,482 issued to Stewart, etal).

“Specifically” or “selectively” binds, when referring to aligand/receptor, antibody/antigen, or other binding pair, indicates abinding reaction which is determinative of the presence of the proteinin a heterogeneous population of proteins and other biologics. Underdesignated conditions, a specified ligand binds to a particular receptorand does not bind in a significant amount to other proteins present inthe sample. The antibody, or binding composition derived from theantigen-binding site of an antibody, of the contemplated method binds toits antigen, or a variant or mutein thereof, with an affinity or bindingconstant that is at least two fold greater, preferably at least tentimes greater, more preferably at least 20-times greater, and mostpreferably at least 100-times greater than the affinity with any otherantibody, or binding composition derived thereof. In a preferredembodiment the antibody will have an affinity that is greater than about10⁹ liters/mol, as determined, e.g., by Scatchard analysis (Munsen, etal. (1980) Analyt. Biochem. 107:220-239).

“Treatment,” as it applies to a human, veterinary, or research subject,refers to therapeutic treatment, prophylactic or preventative measures,to research and diagnostic applications. “Treatment” as it applies to ahuman, veterinary, or research subject, or cell, tissue, or organ,encompasses contact of a IL-17 or IL-23 antagonist to a human or animalsubject, or to a cell, tissue, physiological compartment, orphysiological fluid. “Treatment of a cell, tissue, organ, or subject”encompasses situations where it has not been demonstrated that theantagonist of IL-17 or IL-23 has contacted their respective receptors,or a cell expressing these receptors.

“Therapeutically effective amount” of a therapeutic agent is defined asan amount of each active component of the pharmaceutical formulationthat is sufficient to show a meaningful patient benefit, i.e., to causea decrease in, amelioration of, or prevention of the symptoms of thecondition being treated. When the pharmaceutical formulation comprises adiagnostic agent, “a therapeutically effective amount” is defined as anamount that is sufficient to produce a signal, image, or otherdiagnostic parameter that facilitates diagnosis. Effective amounts ofthe pharmaceutical formulation will vary according to factors such asthe degree of susceptibility of the individual, the age, gender, andweight of the individual, and idiosyncratic responses of the individual(see, e.g., U.S. Pat. No. 5,888,530).

II. General.

Mammalian skin consists of dermal (inner) and epidermal (outer) layers.The epidermis is made almost entirely of keratinocytes (95%) with othercell types including Langerhans cells and melanocytes. The epidermis israpidly growing, turning over every seven days in the mouse. Inpsoriasis, this turnover is shortened to 3-5 days as a result ofkeratinocyte hyperproliferation.

The present invention is based, in part, upon the discovery thatnon-lesional psoriatic tissues expresses IL-17A mRNA at significantlyelevated levels when compared to normal or non-psoriatic tissue. A humaninflammatory skin panel was analyzed by real-time quantitative PCR(TaqMan® real-time quantitative PCR). The skin panel included normal,non-lesional and lesional psoriatic skin tissue, and lesional andnon-lesional atopic dermatitis skin samples. Non-lesional and lesionalpsoriasis tissue was derived from patient samples with PASI (psoriasisarea and severity index) scores between 9-20.75. Non-lesional andlesional atopic dermatitis tissue was derived from patient samples withEASI (eczema area and severity index) scores between 1 .85-35.95.

The results of the PCR analysis were normalized to ubiquitin andKruskal-Wallis statistical analysis was performed on log transformeddata (median method). Normal skin tissue samples had an average IL-17AmRNA expression value of 0.42, while the psoriatic non-lesional tissue,psoriatic lesional tissue, non-lesional atopic dermatitis tissue, andlesional atopic dermatitis tissue had average IL-17A expression valuesof 5.19 (12.3 fold higher), 25.7 (61.2 fold higher), 0.72 (1.7 foldhigher) and 2.1 (5 fold higher), respectively, when compared to normalskin, as described in Table 1 (L=Lesional, NL=Nonlesional, AD=AtopicDermatitis).

TABLE 1 IL-17A Normalized Values Normal skin Psoriasis NL Psoriasis L ADNL AD L 0.13 0.08 0.06 0.12 0.11 4.95 16.41 55.06 0.19 0.12 0.95 19.0046.43 0.08 0.12 0.09 18.13 27.84 0.07 1.53 0.08 0.89 13.35 0.16 0.080.10 0.08 4.69 0.11 0.07 0.06 0.66 5.53 0.09 1.31 0.06 2.37 10.43 0.100.08 1.39 1.79 22.67 0.18 1.46 0.07 3.84 24.76 1.55 6.00 0.09 6.26 51.921.58 0.54 0.76 9.39 49.53 0.16 0.09 0.06 0.09 16.50 0.53 9.01 1.53 0.8722.67 1.29 2.07 0.08 0.05 23.21 0.95 1.46 0.11 1.02 20.51 0.09 0.53 0.200.08 11.26 0.39 3.16 0.11 5.25 6.44 4.29 14.59 0.09 30.77 39.85 0.071.29 0.18 1.05 9.78 0.08 0.05 0.06 18.34 1.07 3.62 0.07 0.10 41.04 0.100.87 0.15 0.84 49.24 0.08 0.09 0.10 0.75 52.22 0.16 0.11 1.54 4.92 19.451.33 2.10 0.12 0.09 1.60 0.09 0.06 0.92 0.11 2.58 5.80 0.95 0.93 2.680.05 3.13 1.62 0.07 0.06 0.05 0.08 0.10 Average 0.42 5.19 25.71 0.722.10 Fold increase over 12.37 61.22 1.72 5.01 normal Median 0.10 1.0322.67 0.16 1.30 Fold increase over 10.84 238.01 1.66 13.68 normal

IL-17F mRNA expression was similarly analyzed. The results of the PCRanalysis were normalized to ubiquitin and Kruskal-Wallis statisticalanalysis was performed on log transformed data (median method). Normalskin tissue samples had an average IL-17F mRNA expression value of 0.90,while the psoriatic non-lesional tissue, psoriatic lesional tissue,non-lesional atopic dermatitis tissue, and lesional atopic dermatitistissue had average IL-17F expression values of 9.39 (10.43 fold higher),55.85 (62.05 fold higher), 1.39 (1.54 fold higher) and 4.13 (4.59 foldhigher), respectively, when compared to normal skin, as described inTable 2 (L=Lesional, NL=Nonlesional, AD=Atopic Dermatitis).

TABLE 2 IL-17F Normalized Values Normal skin Psoriasis NL Psoriasis L ADNL AD L 0.15 0.15 1.71 0.19 1.41 3.22 23.90 67.24 4.72 3.28 1.49 12.3061.57 0.09 0.18 0.27 33.80 64.53 1.89 3.93 0.11 2.59 79.74 0.16 0.090.11 0.13 21.50 3.13 1.19 1.12 0.25 20.51 0.11 0.13 0.08 20.03 36.490.10 2.30 4.22 0.09 85.06 0.24 4.80 0.08 0.17 44.56 0.21 11.13 3.30 3.5482.60 2.17 4.77 2.64 37.14 137.74 0.17 0.09 0.84 0.14 27.52 2.10 7.382.53 1.85 65.68 2.25 7.20 2.58 1.35 21.62 1.79 18.24 0.10 0.15 22.010.16 1.12 0.93 0.08 64.91 0.22 10.19 0.31 13.43 27.20 7.33 38.92 0.8050.12 68.44 0.10 0.12 0.30 7.08 71.73 0.09 0.12 0.13 86.58 1.40 1.170.10 0.17 49.82 0.09 0.09 0.22 0.12 47.54 1.29 0.12 0.12 5.40 106.980.16 1.48 1.16 11.39 33.02 0.11 0.97 0.13 0.14 0.15 0.10 1.62 0.08 0.141.37 2.20 0.77 4.37 0.11 0.16 3.89 0.90 2.83 0.13 0.09 0.09 0.13 Average0.90 9.39 55.85 1.39 4.13 Fold increase 10.43 62.05 1.54 4.59 overnormal Median 0.22 2.22 61.57 0.23 1.18 Fold increase 10.19 282.24 1.045.40 over normal

IL19 mRNA expression was also analyzed. The results of the PCR analysiswere normalized to ubiquitin and Kruskal-Wallis statistical analysis wasperformed on log transformed data (median method). Normal skin tissuesamples had an average IL-19 mRNA expression value of 0.75, while thepsoriatic non-lesional tissue, psoriatic lesional tissue non-lesionalatopic dermatitis tissue, and lesional atopic dermatitis tissue hadaverage IL-19 expression values of 23.44 (31.11 fold higher), 199.40(264.66 fold higher), 9.65 (12.80 fold higher) and 31.55 (41.88 foldhigher), respectively, when compared to normal skin, as described inTable 3 (L=Lesional, NL=Nonlesional, AD=Atopic Dermatitis).

TABLE 3 IL-19 Normalized Values Normal skin Psoriasis NL Psoriasis L ADNL AD L 0.13 3.13 15.47 1.16 5.60 19.68 65.68 192.56 0.19 0.12 1.8040.09 126.86 0.08 1.58 0.09 66.45 133.75 0.72 2.01 0.08 2.28 44.30 0.162.90 0.10 0.84 63.78 0.11 1.09 0.06 0.06 9.17 0.09 0.07 0.06 8.25 30.230.10 0.08 0.06 2.70 588.28 2.97 11.87 0.07 14.16 41.77 0.49 13.04 1.0213.75 784.64 71.73 348.65 0.10 40.56 212.80 8.85 14.50 0.06 4.15 92.366.95 20.51 0.14 17.40 143.53 123.19 282.16 0.08 10.56 97.95 0.16 0.910.11 5.66 39.15 4.83 6.48 0.20 0.99 116.84 0.39 1.36 0.11 11.46 4.489.44 25.80 0.09 163.34 171.20 3.75 4.53 0.18 14.76 63.78 0.08 0.05 0.061.53 129.88 7.38 24.32 0.07 0.07 856.96 2.36 4.64 0.15 7.46 473.30 0.088.30 0.10 67.24 354.86 0.63 27.84 0.07 197.14 4.61 4.48 0.12 0.98 0.120.09 0.06 0.69 0.11 1.85 13.20 0.07 3.38 6.83 0.05 32.63 112.79 0.070.06 0.05 0.97 0.10 Average 0.75 23.44 199.40 6.65 31.55 Fold increase31.11 264.66 12.80 41.88 over normal Median 0.09 9.40 126.86 1.07 5.12Fold increase 99.31 1339.95 11.27 54.03 over normal

IL-17A, first isolated from an activated rodent T-cell hybridoma, wasinitially named CTLA-8 (see, e.g., Rouvier, et al. (1993) J. Immunol.150:5445-5456). The cytokine had substantial homology (58%) to anopen-reading frame in Herpesvirus samiri. Initial biologicalcharacterization found that IL-17 could promote the production of otherinflammatory cytokines and chemokines such as IL-6, IL-8, and G-CSF fromepithelial, endothelial, and fibroblast cells (see, e.g., Yao, et al.(1995) Immunity 3:811-821; Kennedy, et al. (1996) J. Interferon CytokineRes. 16:611-617; and Fossiez, et al. (1996) J. Exp. Med. 183:2593-2603).IL-17 has been associated in various inflammatory and proliferativedisorders, including rheumatoid arthritis, airway inflammation,transplant rejection, systemic sclerosis, psoriasis, and tumor growth(see, e.g., Aggarwal and Gurney (2002) J. Leukocyte Biology 71:1-8).

IL-17F has high sequence identity to IL-17A. However, the level ofexpression of IL-17F (also known as ML-1) is low compared to that ofIL-17A and the expression patterns for IL-17F are quite distinct fromthose of IL-17A. RT-PCR results indicate that IL-17F is expressed byactivated CD4+T cells and activated monocytes (see, e.g., Starnes, etal. (2001) J. Immunol. 167:4137-4140). IL-17F expression and activityhas been linked to pulmonary epithelial cells as well angiogenesis (see,e.g., Starnes, et al. supra; Kawaguchi, et al. (2001) J. Immunol.167:4430-4435; and Kawaguchi, et al. (2004) J. Allergy Clin. Immunol.114:1265-1273). The present invention reveals that expression of IL-17Fin lesional and non-lesional psoriasis and atopic dermatitis mirrors theexpression of IL-17A in the same tissues (see, e.g., Tables 1 and 2).Thus, using one or both of the IL-17 family member expression levelsshould provide useful information in predicting the outbreak of thesetwo inflammatory skin disorders. Also antagonizing one or both of thesecytokines should provide preventative therapy to avoid outbreaks ofthese disorders.

IL-19 is a member of a novel family of IL-10-related cytokines thatconsists of IL-19, IL-20, and IL-24. Monocytes are the majorhematopoietic source of IL-19 (Nagalakshmi et al, (2004) IntImmunopharmacol 4:577-592). Monocytes also respond to IL-19 by producingIL-6 and TNF (Liao et al, (2002) J Immunol 169:4288-4297). IL-19 bindsto the heterodimeric receptor IL-20R1/IL-20R2 (Parrish-Novak (2002), J.Biol Chem 277: 47517-47523). These receptor subunits are highlyexpressed in epithelial cells, including keratinocytes (Wang et al(2002) J Biol Chem 277:7341-7347; Blumberg et al. (2001) Cell 104:9-19),and are elevated in human psoriatic skin (Romer et al (2003) J InvestDermatol 6:1306-1311), suggesting a possible role in psoriasispathogenesis. IL-19 mRNA is expressed by keratinocytes in humanpsoriatic lesions and disappears after treatment with cyclosporin orcalcipotriol (Romer et al, supra). IL-19 mRNA is also reduced inpsoriatic patients after IL-4 therapy (Ghoreschi et al (2003) Nat Med9:40-46). Elevated IL-19 levels in serum has also been associated withasthma (Liao et al (2004) J Immunol. 173:6712-6718).

IL-23 is a heterodimeric cytokine composed of one unique subunit, p19(also known as IL-B30, IL-30) in association with the p40 subunit fromIL-12 (see, e.g., Oppmann, et al. (2000) Immunity 13:715-725).Transgenic overexpression of IL-23p19 was shown to be sufficient for theinduction of systemic inflammation and premature death, however, thiseffect appeared to be IFNγ-independent. Thus, suggesting that IL-23 haseffects independent and substantially different from IL-12 (see, e.g.,Wiekowski, et al. (2001) J. Immunol. 166:7563-7570). Recent studies haveshown that IL-23 may act to induce a distinct T cell activation stateleading to the production of IL-17 (see, Aggarwal, et al. (2003) J.Biol. Chem. 278:1910-1914). Therefore, antagonizing IL-17 and/or IL-23should be efficatious in the prevention of cutaneous inflammation, inparticular, psoriasis.

IL-23 gene expression is increased in human psoriasis (Lee et al, (2004)J Exp Med 199:125-130). To investigate whether IL-23 may play a role indisease pathogenesis, we injected 1 μg IL-23 intradermally daily intothree different wild-type mouse strains (C57B1/6, 129SvEv, and B6×129)and examined its effect on skin histologically and by gene expression.IL-23 induced clinical and histological changes in mouse skin that bearssome similarities to human psoriatic lesions, Table 4.

TABLE 4 Comparison of clinical and histological features of humanpsoriasis with IL-23-treated mouse skin. IL-23-treated Human wild-typepsoriasis skin Clinical Erythematous yes yes Well-demarcated from yes nosymptomless skin White silvery scale yes no, although small areas offlaky skin Histological Acanthosis yes yes Parakeratosis yes yesElongation of rete ridges yes no Intracorneal pustules yes yes (Munromicroabscesses) Recruitment to dermis of- CD4+ lymphocytes yes yesdendritic cells yes yes macrophages yes yes neutrophils yes yes mastcells yes no CD8+ cells in epidermis yes no Prominent dermal yes yespapillary blood vessels

IL-23 induced the expression of IL-17, IL-17F, and IL-19 in comparisonto vehicle control suggesting that these cytokines may form part of animportant pathway whose dysregulation may contribute to psoriasispathogenesis, Table 5. Antagonism of either of these cytokines maytherefore be efficacious in the treatment of cutaneous inflammatorydiseases such as psoriasis.

TABLE 5 IL-23 induces IL-17A, IL-17F, and IL-19 gene expression in mice.Values are relative to ubiquitin and represent pooled mRNA (n = 4) fromthree different wild-type strains. Vehicle Avg. IL-23 Avg Day 1 IL-17A1.42, 9.43, 0.07 3.64 47.86, 48.71, 18.62 38.40 IL-17F 1.2, 1.02, 0.050.76 184.73, 91.21, 66.72 114.22 IL-19 0.89, 3.16, 0.96 1.67 12.9,16.07, 10.04 13.00 Day 4 IL-17A 2.76, 5.81, 0.23 2.93 23.36, 44.19, 4.2723.94 IL-17F 5.53, 1.93, 0.06 2.51 43.24, 38.10, 22.76 34.70 IL-19 0.97,2.82, 4.89 2.89 56.51, 58.22, 109.36 74.70III. Binding Compositions.

Binding compositions provided by the methods of the present inventioninclude reagents such as IL-17, IL-19, and IL-23, soluble receptors, andantibodies, as well as nucleic acids encoding these reagents.

Monoclonal, polyclonal, and humanized antibodies can be prepared (see,e.g., Sheperd and Dean (eds.) (2000) Monoclonal Antibodies, Oxford Univ.Press, New York, N.Y.; Kontermann and Dubel (eds.) (2001) AntibodyEngineering, Springer-Verlag, New York; Harlow and Lane (1988)Antibodies A Laboratory Manual, Cold Spring Harbor Laboratory Press,Cold Spring Harbor, N.Y., pp. 139-243; Carpenter, et al. (2000) J.Immunol. 165:6205; He, et al. (1998) J. Immunol. 160:1029; Tang, et al.(1999) J. Biol. Chem. 274:27371-27378; Baca, et al. (1997) J. Biol.Chem. 272:10678-10684; Chothia, et al. (1989) Nature 342:877-883; Footeand Winter (1992) J. Mol. Biol. 224:487-499; U.S. Pat. No. 6,329,511issued to Vasquez, et al.).

An alternative to humanization is to use human antibody librariesdisplayed on phage or human antibody libraries in transgenic mice(Vaughan, et al. (1996) Nature Biotechnol. 14:309-314; Barbas (1995)Nature Medicine 1:837-839; Mendez, et al. (1997) Nature Genetics15:146-156; Hoogenboom and Chames (2000) Immunol. Today 21:371-377;Barbas, et al. (2001) Phage Display:A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y.; Kay, et al. (1996)Phage Display of Peptides and Proteins:A Laboratory Manual, AcademicPress, San Diego, Calif.; de Bruin, et al. (1999) Nature Biotechnol.17:397-399).

Single chain antibodies and diabodies are described (see, e.g., Malecki,et al. (2002) Proc. Natl. Acad. Sci. USA 99:213-218; Conrath, et al.(2001) J. Biol. Chem. 276:7346-7350; Desmyter, et al. (2001) J. Biol.Chem. 276:26285-26290; Hudson and Kortt (1999) J. Immunol. Methods231:177-189; and U.S. Pat. No. 4,946,778). Bifunctional antibodies areprovided (see, e.g., Mack, et al. (1995) Proc. Natl. Acad. Sci. USA92:7021-7025; Carter (2001) J. Immunol. Methods 248:7-15; Volkel, et al.(2001) Protein Engineering 14:815-823; Segal, et al. (2001) J. Immunol.Methods 248:1-6; Brennan, et al. (1985) Science 229:81-83; Raso, et al.(1997) J. Biol. Chem. 272:27623; Morrison (1985) Science 229:1202-1207;Traunecker, et al. (1991) EMBO J. 10:3655-3659; and U.S. Pat. Nos.5,932,448, 5,532,210, and 6,129,914).

The present invention provides a bispecific antibody that can bindspecifically to at least two cytokines chosen from IL-17, IL-19, orIL-23, or receptors, thereof. (see, e.g., Azzoni, et al. (1998) J.Immunol. 161:3493; Kita, et al. (1999) J. Immunol. 162:6901; Merchant,et al. (2000) J. Biol. Chem. 74:9115; Pandey, et al. (2000) J. Biol.Chem. 275:38633; Zheng, et al. (2001) J. Biol Chem. 276:12999; Propst,et al. (2000) J. Immunol. 165:2214; Long (1999) Ann. Rev. Immunol.17:875).

Purification of antigen is not necessary for the generation ofantibodies. Animals can be immunized with cells bearing the antigen ofinterest. Splenocytes can then be isolated from the immunized animals,and the splenocytes can fused with a myeloma cell line to produce ahybridoma (see, e.g., Meyaard, et al. (1997) Immunity 7:283-290; Wright,et al. (2000) Immunity 13:233-242; Preston, et al., supra; Kaithamana,et al. (1999) J. Immunol. 163:5157-5164).

Antibodies will usually bind with at least a K_(D) of about 10⁻³ M, moreusually at least 10⁻⁶ M, typically at least 10⁻⁷ M, more typically atleast 10⁻⁸ M, preferably at least about 10⁻⁹ M, and more preferably atleast 10⁻¹⁰ M, and most preferably at least 10⁻¹¹ M (see, e.g., Presta,et al. (2001) Thromb. Haemost. 85:379-389; Yang, et al. (2001) Crit.Rev. Oncol. Hematol. 38:17-23; Carnahan, et al. (2003) Clin. Cancer Res.(Suppl.) 9:3982s-3990s).

Polypeptides, antibodies, and nucleic acids, can be conjugated, e.g., tosmall drug molecules, enzymes, liposomes, polyethylene glycol (PEG), orfusion protein antibodies. Antibodies are useful for diagnostic or kitpurposes, and include antibodies coupled, e.g., to dyes, radioisotopes,enzymes, or metals, e.g., colloidal gold (see, e.g., Le Doussal, et al.(1991) J. Immunol. 146:169-175; Gibellini, et al. (1998) J. Immunol.160:3891-3898; Hsing and Bishop (1999) J. Immunol. 162:2804-2811;Everts, et al. (2002) J. Immunol. 168:883-889).

The invention also provides binding compositions for use as anti-sensenucleic acids or for small interference RNA (siRNA) (see, e.g., Arenzand Schepers (2003) Naturwissenschaften 90:345-359; Sazani and Kole(2003) J. Clin. Invest. 112:481-486; Pirollo, et al. (2003) Pharmacol.Therapeutics 99:55-77; Wang, et al. (2003) Antisense Nucl. Acid DrugDevel. 13:169-189; Cheng, et al. (2003) Mol. Genet. Metab. 80:121-128;Kittler and Buchholz (2003) Semin. Cancer Biol. 13:259-265).

IV. Purification and Modification of Polypeptides and Nucleic Acids.

Polypeptides, e.g., antigens, antibodies, and antibody fragments, andnucleic acids for use in the contemplated method, can be purified bymethods that are established in the art. Purification can involvehomogenization of cells or tissues, immunoprecipitation, andchromatography. Stability during purification or storage can beenhanced, e.g., by anti-protease agents, anti-oxidants, ionic andnon-ionic detergents, and solvents, such as glycerol ordimethylsulfoxide.

Modification of, e.g., peptides, polypeptides, and nucleic acids,includes epitope tags, fluorescent or radioactive groups,monosaccharides or oligosaccharides, sulfate or phosphate groups,C-terminal amides, acetylated and esterified N-groups, acylation, e.g.,fatty acid, intrachain cleaved peptide bonds, and deamidation products(see, e.g., Johnson, et al. (1989) J. Biol. Chem. 264:14262-14271;Young, et al. (2001) J. Biol. Chem. 276:37161-37165). Glycosylationdepends upon the nature of the recombinant host organism employed orphysiological state (see, e.g., Jefferis (2001) BioPharm 14:19-27;Mimura, et al. (2001) J. Biol. Chem. 276:45539-45547; Axford (1999)Biochim. Biophys. Acta 1:219-229; Malhotra, et al. (1995) NatureMedicine 1:237-243).

V. Therapeutic Compositions and Methods.

To prepare pharmaceutical or sterile compositions including anantagonist of IL-17 or IL-23, the reagents is mixed with apharmaceutically acceptable carrier or excipient. Formulations oftherapeutic, prophylactic, and diagnostic agents can be prepared bymixing with physiologically acceptable carriers, excipients, orstabilizers in the form of, e.g., lyophilized powders, slurries, aqueoussolutions, lotions, or suspensions (see, e.g., Hardman, et al. (2001)Goodman and Gilman's The Pharmacological Basis of Therapeutics,McGraw-Hill, New York, N.Y.; Gennaro (2000) Remington: The Science andPractice of Pharmacy, Lippincott, Williams, and Wilkins, New York, N.Y.;Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: ParenteralMedications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990)Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, etal. (eds.) (1990) Pharmaceutical Dosage Forms: Disperse Systems, MarcelDekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety,Marcel Dekker, Inc., New York, N.Y.).

Selecting an administration regimen for a prophylactic or therapeuticdepends on several factors, including the serum or tissue turnover rateof the entity, the level of symptoms, the immunogenicity of the entity,and the accessibility of the target cells in the biological matrix.Preferably, an administration regimen maximizes the amount oftherapeutic delivered to the patient consistent with an acceptable levelof side effects. Accordingly, the amount of biologic delivered dependsin part on the particular entity and the severity of the condition beingtreated. Guidance in selecting appropriate doses of antibodies,cytokines, and small molecules are available (see, e.g., Wawrzynczak(1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK;Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis,Marcel Dekker, New York, N.Y.; Bach (ed.) (1993) Monoclonal Antibodiesand Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York,N.Y.; Baert, et al. (2003) New Engl. J. Med. 348:601-608; Milgrom, etal. (1999) New Engl. J. Med. 341:1966-1973; Slamon, et al. (2001) NewEngl. J. Med. 344:783-792; Beniaminovitz, et al. (2000) New Engl. J.Med. 342:613-619; Ghosh, et al. (2003) New Engl. J. Med. 348:24-32;Lipsky, et al. (2000) New Engl. J. Med. 343:1594-1602).

Antibodies, antibody fragments, and cytokines can be provided bycontinuous infusion, or by doses at intervals of, e.g., one day, oneweek, or 1-7 times per week. Doses may be provided intravenously,subcutaneously, topically, orally, nasally, rectally, intramuscular,intracerebrally, or by inhalation. A preferred dose protocol is oneinvolving the maximal dose or dose frequency that avoids significantundesirable side effects. A total weekly dose is generally at least 0.05μg/kg body weight, more generally at least 0.2 μg/kg, most generally atleast 0.5 μg/kg, typically at least 1 μg/kg, more typically at least 10μg/kg, most typically at least 100 μg/kg, preferably at least 0.2 mg/kg,more preferably at least 1.0 mg/kg, most preferably at least 2.0 mg/kg,optimally at least 10 mg/kg, more optimally at least 25 mg/kg, and mostoptimally at least 50 mg/kg (see, e.g., Yang, et al. (2003) New Engl. J.Med. 349:427-434; Herold, et al. (2002) New Engl. J. Med. 346:1692-1698;Liu, et al. (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji,et al. (20003) Cancer Immunol. Immunother. 52:133-144). The desired doseof a small molecule therapeutic, e.g., a peptide mimetic, naturalproduct, or organic chemical, is about the same as for an antibody orpolypeptide, on a moles/kg body weight basis. The desired plasmaconcentration of a small molecule therapeutic is about the same as foran antibody, on a moles/kg body weight basis.

An effective amount for a particular patient may vary depending onfactors such as the condition being treated, the overall health of thepatient, the method route and dose of administration and the severity ofside affects, see, e.g., Maynard, et al. (1996) A Handbook of SOPs forGood Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001)Good Laboratory and Good Clinical Practice, Urch Publ., London, UK.

Typical veterinary, experimental, or research subjects include monkeys,dogs, cats, rats, mice, rabbits, guinea pigs, horses, and humans.

Determination of the appropriate dose is made by the clinician, e.g.,using parameters or factors known or suspected in the art to affecttreatment or predicted to affect treatment. Generally, the dose beginswith an amount somewhat less than the optimum dose and it is increasedby small increments thereafter until the desired or optimum effect isachieved relative to any negative side effects. Important diagnosticmeasures include those of symptoms of, e.g., the inflammation or levelof inflammatory cytokines produced. Preferably, a biologic that will beused is derived from the same species as the animal targeted fortreatment, thereby minimizing a humoral response to the reagent.

Methods for co-administration or treatment with a second therapeuticagent, e.g., a cytokine, steroid, chemotherapeutic agent, antibiotic, orradiation, are well known in the art (see, e.g., Hardman, et al. (eds.)(2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics,10^(th) ed., McGraw-Hill, New York, N.Y.; Poole and Peterson (eds.)(2001) Pharmacotherapeutics for Advanced Practice:A Practical Approach,Lippincott, Williams & Wilkins, Phila., Pa.; Chabner and Longo (eds.)(2001) Cancer Chemotherapy and Biotherapy, Lippincott, Williams &Wilkins, Phila., Pa.). An effective amount of therapeutic will decreasethe symptoms typically by at least 10%; usually by at least 20%;preferably at least about 30%; more preferably at least 40%, and mostpreferably by at least 50%.

The route of administration is by, e.g., topical or cutaneousapplication, injection or infusion by intravenous, intraperitoneal,intracerebral, intramuscular, intraocular, intraarterial,intracerebrospinal, intralesional, or pulmonary routes, or by sustainedrelease systems or an implant (see, e.g., Sidman et al. (1983)Biopolymers 22:547-556; Langer, et al. (1981) J. Biomed. Mater. Res.15:167-277; Langer (1982) Chem. Tech. 12:98-105; Epstein, et al. (1985)Proc. Natl. Acad. Sci. USA 82:3688-3692; Hwang, et al. (1980) Proc.Natl. Acad. Sci. USA 77:4030-4034; U.S. Pat. Nos. 6,350,466 and6,316,024).

VI. Kits.

The present invention diagnostic kits/ Provided are bindingcompositions, including antibodies or antibody fragments, for thedetection of IL-17, IL-19, or IL-23, and metabolites and breakdownproducts thereof, including products resulting from deamidation, limitedproteolytic or hydrolytic cleavage, or disulfide bond oxidation orformation. Typically, the kit will have a compartment containing eithera IL-17, IL-19, or IL-23 polypeptide, or an antigenic fragment thereof,a binding composition thereto, or a nucleic acid, e.g., a nucleic acidprobe or primer, able to hybridize under stringent conditions to anucleic acid encoding IL-17, IL-19, or IL-23.

The kit can comprise, e.g., a reagent and a compartment, a reagent andinstructions for use, or a reagent with a compartment and instructionsfor use. The reagent can comprise full length IL-17, IL-19, or IL-23polypeptide, or an antigenic fragment thereof, a binding composition, ora nucleic acid. A kit for determining the binding of a test compound,e.g., acquired from a biological sample or from a chemical library, cancomprise a control compound, a labeled compound, and a method forseparating free labeled compound from bound labeled compound.

Conditions enabling stringent hybridization of nucleic acid probes orprimers are available (see, e.g., Freeman, et al. (2000) Biotechniques29:1042-1055; de Silva and Wittwer (2000) J. Chromatogr. B. Biomed. Sci.Appl. 741:3-13; Long (1998) Eur. J. Histochem. 42:101-109; Musiani, etal. (1998) Histol. Histopathol. 13:243-248; Gillespie (1990) Vet.Microbiol. 24:217-233; Giulietti, et al. (2001) Methods 25:386-401;Schweitzer and Kingsmore (2001) Curr. Opin. Biotechnol. 12:21-27; Speel,et al. (1999) J. Histochem. Cytochem. 47:281-288; Tsuruoka and Karube(2003) Comb. Chem. High Throughput Screen. 6:225-234; Rose, et al.(2002) Biotechniques 33:54-56).

Diagnostic assays can be used with biological matrices such as livecells, cell extracts, cell lysates, fixed cells, cell cultures, bodilyfluids, or forensic samples. Conjugated antibodies useful for diagnosticor kit purposes, include antibodies coupled to dyes, isotopes, enzymes,and metals (see, e.g., Le Doussal, et al. (1991) New Engl. J. Med.146:169-175; Gibellini, et al. (1998) J. Immunol. 160:3891-3898; Hsingand Bishop (1999) New Engl. J. Med. 162:2804-2811; Everts, et al. (2002)New Engl. J. Med. 168:883-889). Various assay formats exist, such asReal-time PCR, radioimmunoassays (RIA), ELISA, and lab on a chip (U.S.Pat. Nos. 6,176,962 and 6,517,234).

The diagnostic method can comprise contacting a sample from a testsubject with a binding composition that specifically binds to apolypeptide or nucleic acid of IL-17, IL-19, or IL-23. Moreover, thediagnostic method can further comprise contacting the bindingcomposition to a sample derived from a control subject or controlsample, and comparing the binding found with the test subject with thebinding found with the control subject or control sample. A “testsample” can be derived from a skin sample from a subject experiencingpsoriasis, both lesional and non-lesional, while a “control sample” canbe derived from a skin sample from a normal subject, or derived from anon-affected skin sample from the subject experiencing cutaneousinflammation. The subject can be, e.g., human, veterinary, experimental,or agricultural. Derived encompasses a biopsy, sample, extract, or aprocessed, purified, or semi-purified sample or extract.

Alternatively, both test and normal skin samples, as defined above, canbe obtained and subjected to standard mRNA extraction protocols. ThemRNA is subsequently reversed transcribed into ssDNA, which is then usedfor a second DNA strand synthesis. The double strand DNA is then used inreal-time PCR, e.g., TaqMan® real-time Quantitative PCR, reactions. Dataare analyzed as described below.

VII. Uses.

The invention provides methods for the diagnosis or prevention ofcutaneous inflammation, including but not limited to, cicatricialpemphigoid, scleroderma, hidradenitis suppurativa, toxic epidermalnecrolysis, acne, osteitis, graft vs. host disease (GvHD), pyrodermagangrenosum, and Behcet's Syndrome (see, e.g., Willams and Griffiths(2002), supra). The most common form of cutaneous inflammation ispsoriasis.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the inventionsto the specific embodiments.

EXAMPLES

I. General Methods.

Methods for the diagnosis, prevention, and treatment of inflammatoryconditions of the skin in animals and humans are described (see, e.g.,Ackerman (1997) Histological Diagnosis of Inflammatory Skin Disease,2^(nd) ed., Lippincott, Williams, and Wilkins, New York, N.Y.; Gallin,et al. (1999) Inflammation:Basic Principles and Clinical Correlates,3^(rd) ed., Lippincott, Williams, and Wilkins, New York, N.Y.; Parnham,et al. (1991) Drugs in Inflammation (Agents and Actions Suppl., Vol.32), Springer Verlag, Inc., New York, N.Y.; Chan (ed.) (2003) AnimalModels of Human Inflammatory Skin Diseases, CRC Press, Boca Raton, Fla.;Kownatzki and Norgauer (eds.) (1998) Chemokines and Skin, BirkhauserVerlag, Basel, Switzerland; Kanitakis, et al. (eds.) (1999) DiagnosticImmunohistochemistry of the Skin, Lippincott, Williams, and Wilkins, NewYork, N.Y.).

Animal models of cutaneous inflammation, and related methods, areavailable. These methods include use of skin grafts, skin graftsinjected with immune cells, subcutaneous injection of immune cells, anduse of animals such as various mouse models of psoriasis, in particularxenotransplatation models (see, e.g., Kruger, et al. (1981) J. Clin.Invest., 68:1548-1577; Nickoloff, et al. (1995) Am. J. Pathol.146:580-588; and Schön (1999) J. Invest. Dermatol. 112:405-410).

Standard methods in molecular biology are described (Maniatis, et al.(1982) Molecular Cloning, A Laboratory Manual, Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Sambrook and Russell (2001)Molecular Cloning, 3^(rd) ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.; Wu (1993) Recombinant DNA, Vol. 217, AcademicPress, San Diego, Calif.). Standard methods also appear in Ausbel, etal. (2001) Current Protocols in Molecular Biology, Vols. 1-4, John Wileyand Sons, Inc. New York, N.Y., which describes cloning in bacterialcells and DNA mutagenesis (Vol. 1), cloning in mammalian cells and yeast(Vol. 2), glycoconjugates and protein expression (Vol. 3), andbioinformatics (Vol. 4).

Methods for protein purification including immunoprecipitation,chromatography, electrophoresis, centrifugation, and crystallization aredescribed (Coligan, et al. (2000) Current Protocols in Protein Science,Vol. 1, John Wiley and Sons, Inc., New York). Chemical analysis,chemical modification, post-translational modification, production offusion proteins, glycosylation of proteins are described (see, e.g.,Coligan, et al. (2000) Current Protocols in Protein Science, Vol. 2,John Wiley and Sons, Inc., New York; Ausubel, et al. (2001) CurrentProtocols in Molecular Biology, Vol. 3, John Wiley and Sons, Inc., NY,N.Y., pp. 16.0.5-16.22.17; Sigma-Aldrich, Co. (2001) Products for LifeScience Research, St. Louis, Mo.; pp. 45-89; Amersham Pharmacia Biotech(2001) BioDirectory, Piscataway, N.J., pp. 384-391). Production,purification, and fragmentation of polyclonal and monoclonal antibodiesis described (Coligan, et al. (2001) Current Protcols in Immunology,Vol. 1, John Wiley and Sons, Inc., New York; Harlow and Lane (1999)Using Antibodies, Cold Spring Harbor Laboratory Press, Cold SpringHarbor, N.Y.; Harlow and Lane, supra). Standard techniques forcharacterizing ligand/receptor interactions are available (see, e.g.,Coligan, et al. (2001) Current Protcols in Immunology, Vol. 4, JohnWiley, Inc., New York).

Standard techniques in cell and tissue culture are described (see, e.g.,Freshney (2000) Culture of Animal Cells: A Manual of Basic Technique,4^(th) ed., Wiley-Liss, Hoboken, N.J.; Masters (ed.) (2000) Animal CellCulture: A Practical Approach, 3^(rd) ed., Oxford Univ. Press, Oxford,UK; Doyle, et al. (eds.) (1994) Cell and Tissue Culture: LaboratoryProcedures, John Wiley and Sons, NY; Melamed, et al. (1990) FlowCytometry and Sorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988)Practical Flow Cytometry Liss, New York, N.Y.; Robinson, et al. (1 993)Handbook of Flow Cytometry Methods, Wiley-Liss, New York, N.Y.).

Software packages for determining, e.g., antigenic fragments, signal andleader sequences, protein folding, and functional domains, areavailable. See, e.g., Vector NTI® Suite (Informax, Inc., Bethesda, Md.);GCG Wisconsin Package (Accelrys, Inc., San Diego, Calif.), and DeCypher®(TimeLogic Corp., Crystal Bay, Nev.); Menne, et al. (2000)Bioinformatics 16:741-742. Public sequence databases were also used,e.g., from GenBank and others.

II. Real-time PCR

Total RNA from homogenized tissue samples (see below) was extracted andreverse transcribed as previously described (see, e.g., Homey, et al.(2000) J. Immunol. 164:3465-3470). Complementary DNA was quantitativelyanalyzed for expression of IL-17 by the fluorgenic 5′-nuclease PCR assay(see, e.g., Holland, et al. (1991) Proc. Natl. Acad. Sci. 88:7276-7280).Specific primers used were as follows:

Human IL-17A Forward Primer 5′-CAACCGATCCACCTCACCTT-3′ SEQ ID NO:1 HumanIL-17A Reverse Primer 5′-GGCACTTTGCCTCCCAGAT-3′ SEQ ID NO:2 Human IL-17FForward Primer: 5′-TGCCAGGAGGTAGTATGAAGCTT-3′ SEQ ID NO:3 Human IL-17FReverse Primer: 5′-ATGCAGCCCAAGTTCCTACACT-3′ SEQ ID NO:4 Human IL-19Forward Primer: 5′-TTGGCTCCTGGGTACAATACTG-3′ SEQ ID NO:5 Human IL-19Reverse Primer: 5′-TCCTTAGCTTGGATGGCTCTTT-3′ SEQ ID NO:6

IL-17 or IL-19 specific PCR products were continuously measured by meansof an ABI PRISM 7700 Sequence Detection System (Applied Biosystems)during 40 cycles. Values were normalized to ubiquitin. Log-transformeddata was subjected to Kruskal-Wallis statistical analysis (medianmethod). The expression level (log transformed) corresponds to theamount of IL-17 or IL-19 expressed in the tissue sample, such that thehigher the expression level (log transformed), the greater the amount ofIL-17 or IL-19 expressed in the tissue sample.

III. Human Inflammatory Skin Disease Panel

The human inflammatory skin disease panel included normal skin,non-lesional and lesional skin from psoriatic and atopic dermatitispatients. The panel included 35 normal skin samples (15 from autopsydonors and 20 from normal donors in clinical trial setting, see below),24 non-lesional psoriasis skin samples, 25 lesional psoriasis skinsamples, 30 non-lesional atopic dermatitis skin samples, and 30 lesionalatopic dermatitis skin samples. Two 4mm punch biopsies were taken fromeach patient. Non-lesional psoriatic and atopic dermatitis skin sampleswere taken from sites distal to a psoriatic or atopic dermatitis lesion.Samples were obtained in a clinical trial setting at Stanford UniversityDermatology Department. Autopsy donor materials were obtained fromZoion. The study was approved by the local ethics committees of therespective institutions.

All non-lesional and lesional patient samples were ranked by severityusing either the PASI (psoriasis area and severity index) score or EASI(eczema area and severity index) score. For psoriasis patients, the PASIscores were in the range of 9-20.75. For atopic dermatitis patients, theEASI scores were in the range of 1.85-35.95. These scores reflected theextent and severity of disease over the patient's body.

IV. Methods for Cell Culture, Histology, and Skin Grafting.

Alternatively, cell lines may be used. Cell lines are cultured inDulbecco's modified Eagle medium (DMEM) (GIBCO BRL, Grand Island, N.Y.)supplemented with 10% fetal bovine serum (FBS). Human keratinocytes maybe derived from newborn human foreskins and are cultured in KeratinocyteSFM (GIBCO BRL; Rheinwald and Green (1975) Cell 6:317-330). Skin isseparated by gently tearing along the cartilage plate and floated on0.5% trypsin (GIBCO BRL) in phosphate buffered saline (PBS) at 37° C.for 45 min. Epidermal sheets are peeled from the dermis, re-suspended in0.05% DNAase (Sigma, St. Louis, Mo.) in PBS containing 10% fetal bovineserum (FBS). Single cell suspension is obtained by vigorous passagethrough a syringe. For reverse transcription polymerase chain reaction(RT-PCR) analysis, cells are cultured in Keratinocyte SFM, and RNA isisolated by standard methodologies.

For flow cytometry, freshly isolated epidermal cells are washed once incold phosphate buffered saline (PBS) and 4×10⁵ cells are stained for 30min at 4° C. with any of the appropriately labeled antibody reagents.Cells are washed twice in cold PBS and analyzed by flow cytometry on aBecton Dickenson FACScan® flow cytometer (San Jose, Calif.).

Many modifications and variations of this invention, as will be apparentto one of ordinary skill in the art, can be made to adapt to aparticular situation, material, composition of matter, process, processstep or steps, to preserve the objective, spirit, and scope of theinvention. All such modifications are intended to be within the scope ofthe claims appended hereto without departing from the spirit and scopeof the invention. The specific embodiments described herein are offeredby way of example only, and the invention is to be limited by the termsof the appended claims, along with the full scope of the equivalents towhich such claims are entitled; and the invention is not to be limitedby the specific embodiments that have been presented herein by way ofexample.

1. A method of evaluating the propensity of a subject to develop aninflammatory skin disorder comprising: a) obtaining a non-lesional skinsample from the subject; b) quantifying the level of IL-17A, IL-17F orIL-19 expression in the skin sample; and c) comparing the level ofIL-17A, IL-17F or IL-19 in the skin sample to the level in normal skinsamples, wherein higher levels of expression of IL-17A, IL-17F or IL-19than normal skin indicate a propensity of the subject to develop aninflammatory skin disorder.
 2. The method of claim 1, wherein theIL-17A, IL-17F or IL-19 expression is mRNA expression.
 3. The method ofclaim 1, wherein the level of IL-17A, IL-17F or IL-19 expression isquantified by real-time PCR.
 4. The method of claim 1, wherein theinflammatory skin disorder is cutaneous inflammation.
 5. The method ofclaim 4, wherein the cutaneous inflammation is psoriasis.
 6. The methodof claim 1, wherein the level of IL-17A or IL-17F expression in the skinsample is at least 5-fold higher than normal skin.
 7. The method ofclaim 1, wherein the level of IL-19 expression in the skin sample is atleast 20-fold higher than normal skin.
 8. The method of claim 1, whereinthe subject has: a) a family history of psoriasis; or b) previouslypresented psoriatic symptoms.
 9. The method of claim 1, wherein thesubject is a human.
 10. The method of claim 1, wherein the level ofIL-17A or IL-17F expression in the skin sample is 2 to 50 fold higherthan normal skin.
 11. The method of claim 10, wherein the level ofIL-17A or IL-17F expression in the skin sample is 5 to 20 fold higherthan normal skin.
 12. The method of claim 1, wherein the level of IL-19expression in the skin sample is 5 to 130 fold higher than normal skin.